Molecular mechanism of microtubule nucleation from gamma ...61 TuRC (Fig. S1E), we showed that γ-TuRC caps the MT minus-end, while only the plus-end 62 polymerizes. Altogether, our

1

Molecular mechanism of microtubule nucleation from gamma-tubulin ring complex 1

2

Akanksha Thawani1, Howard A Stone2, Joshua W Shaevitz3,4, Sabine Petry5,* 3

4

1Department of Chemical and Biological Engineering, Princeton University 5

2Department of Mechanical and Aerospace Engineering, Princeton University 6

3Lewis-Sigler Institute for Integrative Genomics, Princeton University 7

4Department of Physics, Princeton University 8

5Department of Molecular Biology, Princeton University, United States 9

10

* Correspondence to: Sabine Petry ([email protected]) 11

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 23, 2019. ; https://doi.org/10.1101/853010doi: bioRxiv preprint

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Abstract 12

Determining how microtubules (MTs) are nucleated is essential for understanding how the 13

cytoskeleton assembles. Yet, half a century after the discovery of MTs and ab-tubulin subunits 14

and decades after the identification of the γ-tubulin ring complex (γ-TuRC) as the universal MT 15

nucleator, the underlying mechanism largely remains a mystery. Using single molecule studies, 16

we uncover that γ-TuRC nucleates a MT more efficiently than spontaneous assembly. The laterally 17

interacting array of γ-tubulins on γ-TuRC facilitates the lateral association of αβ-tubulins, while 18

longitudinal affinity between γ/αβ-tubulin is surprisingly weak. During nucleation, 3-4 αβ-tubulin 19

dimers bind stochastically to γ-TuRC on average until two of them create a lateral contact and 20

overcome the nucleation barrier. Although γ-TuRC defines the nucleus, XMAP215 significantly 21

increases reaction efficiency by facilitating ab-tubulin incorporation. In sum, we elucidate how 22

MT initiation occurs from γ-TuRC and determine how it is regulated. 23


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Introduction 24

Microtubules (MTs) enable cell division, motility, intracellular organization and transport. MTs 25

were found to consist of αβ-tubulin dimers fifty years ago, yet, how MTs are nucleated in the cell 26

to build the cytoskeleton remains poorly understood1–3. 27

MTs assemble spontaneously from αβ-tubulin subunits in vitro via the cooperative assembly 28

of many tubulin dimers and hence this process displays a kinetic barrier4–8. Consequently, 29

spontaneous MT nucleation is rarely observed in cells9,10. Instead, the major MT nucleator γ-30

tubulin is required in vivo9–11. γ-tubulin forms a 2.2 megadalton, ring-shaped complex with γ-31

tubulin complex proteins (GCPs), known as the γ-Tubulin Ring Complex (γ-TuRC) 12–16. γ-TuRC 32

has been proposed to template the assembly of αβ-tubulin dimers into a ring, resulting in nucleation 33

of a MT15–21. However, kinetic measurements that provide direct evidence for this hypothesis have 34

been lacking and several important questions about how γ-TuRC nucleates MTs have remained 35

unanswered. 36

In the absence of purified g-TuRC and an assay to visualize MT nucleation events from 37

single g-TuRC molecules in real time, recent studies used alternative MT assembly sources, such 38

as spontaneous MT assembly or stabilized MTs with blunt ends hypothesized to resemble the γ-39

TuRC interface. Based on these alternatives, competition between growth and catastrophe of the 40

nascent plus-end was proposed to yield the nucleation barrier in the cell22,23, but this has not been 41

examined with the nucleator g-TuRC. Recently, the MT polymerase XMAP215 was identified as 42

an essential MT nucleation factor in vivo, which synergistically nucleates MTs with γ-TuRC24–26. 43

Yet, the specific roles of XMAP215 and γ-TuRC in MT nucleation have yet to be discovered. 44

To explore the mechanism of MT nucleation, we reconstituted and visualized MT nucleation 45

by γ-TuRC live with single molecule resolution. We uncover the molecular composition of the 46


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MT nucleus, and determine the roles XMAP215 and γ-TuRC in MT nucleation. 47

48


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Results 49

50

Reconstituting and visualizing microtubule nucleation from γ-TuRC 51

To study how γ-TuRC nucleates MTs (Fig. 1A), we purified endogenous γ-TuRC from Xenopus 52

egg extracts and biotinylated the complexes to immobilize them on functionalized glass (Fig. S1A-53

C). Upon perfusing fluorescent αβ-tubulin, we visualized MT nucleation live with total internal 54

reflection fluorescence microscopy (TIRFM). Strikingly, MT nucleation events occurred 55

specifically from single γ-TuRC molecules (Fig. 1B; Fig. S1D and Movie S1-2). Kymographs 56

revealed that attached γ-TuRC assembled ab-tubulin into a MT de novo starting from zero length 57

within the diffraction limit of light microscopy (Fig. 1C), ruling out an alternative model where 58

MTs first spontaneously nucleate and then become stabilized via γ-TuRC. By observing the 59

fiduciary marks on the MT lattice (Fig. 1C) and generating polarity-marked MTs from attached g-60

TuRC (Fig. S1E), we showed that γ-TuRC caps the MT minus-end, while only the plus-end 61

polymerizes. Altogether, our results show that γ-TuRC directly nucleates MTs. 62

63

Defining the microtubule nucleus on γ-TuRC 64

To determine how γ-TuRC nucleates MTs, we measured the kinetics of MT nucleation for a 65

constant density of γ-TuRC and increasing αβ-tubulin concentration (Fig. 1D and Movie S3). 66

Surprisingly, γ-TuRC nucleated MTs starting from 7 µM tubulin (Fig. 1D), which is higher than 67

the minimum tubulin concentration (C*) needed for growth at pre-formed MT plus-ends (C* = 1.4 68

µM, Fig. 1E). Furthermore, the number of MTs nucleated from γ-TuRC increased non-linearly 69

with tubulin concentration as opposed to the linear increase in MT’s growth-speed with tubulin 70

concentration (Fig. 1E). By measuring the number of MTs nucleated over time with varying ab-71


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tubulin concentration (Fig. 1F), we calculated the rate of MT nucleation. The power-law 72

dependence on tubulin concentration (Fig. 1G) yields the number of ab-tubulin dimers, 3.7 ± 0.5, 73

that initiate MT assembly from g-TuRC (Fig. 1G). Thus, the cooperative assembly of 3-4 tubulin 74

subunits on g-TuRC represents the most critical, rate-limiting step in MT nucleation. 75

76

Efficiency of γ-TuRC-mediated nucleation 77

Based on the traditional, fixed, end-point assays for MT nucleation with large error margins, g-78

TuRC was believed to be a poor nucleator14. To measure the efficiency of γ-TuRC-mediated MT 79

nucleation, we compared it with spontaneous MT nucleation in our live TIRFM assay (Fig. 1H). 80

In contrast to γ-TuRC-mediated nucleation, a high concentration of 14 µM tubulin was required 81

for spontaneous assembly of MTs, after which both the plus- and minus-ends polymerize (Fig. 1H, 82

Fig. S1F and Movie S4). The number of MTs assembled as a function of the ab-tubulin 83

concentration displayed a power-law dependence with the exponent of 8 ± 1 (Fig. 1I), indicating 84

a highly cooperative process that requires 8 ab-tubulin dimers in a rate-limiting intermediate, in 85

agreement with previous reports (Fig. 1H schematic, refs 4,8). In conclusion, γ-TuRC nucleates 86

MTs significantly more efficiently (Fig. S1G), because its critical nucleus requires less than half 87

the number of ab-tubulin dimers compared to spontaneous assembly. 88

89

Does γ-TuRC nucleate a microtubule via the blunt plus-end model? 90

It has been widely proposed that the g-tubulin ring on γ-TuRC resembles the blunt plus-end of a 91

MT formed by a ring of ab-tubulins20,22,27. To test this proposition, we generated stabilized MT 92

seeds with blunt ends as described recently22 and observed MT assembly from αβ-tubulin dimers 93

(Fig. 2A). At a minimum concentration of 2.45 µM, approaching the critical concentration needed 94


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for polymerization, a large proportion of pre-formed MT seeds assembled MTs immediately (Fig. 95

S2A-B, Fig. 2A and Movie S5). The measured reaction kinetics (Fig. 2B) as a function of the ab-96

tubulin concentration was used to obtain a power-law of the nucleation rate, 1.2 ± 0.4 (Fig. 2C). 97

This demonstrates that blunt MT seeds assemble tubulin dimers into a lattice in a non-cooperative 98

manner, where a single ab-tubulin dimer suffices to overcome the rate-limiting step resembling 99

the polymerization of a MT. Thus, the kinetics of γ-TuRC-mediated MT nucleation does not 100

resemble a blunt MT plus-end. 101

102

Molecular insight into microtubule nucleation by g-TuRC 103

We hypothesized that γ-tubulin’s binding properties with ab-tubulin at the nucleation interface γ-104

TuRC could provide insight into the mechanism of nucleation. We purified γ-tubulin, which 105

assembles into higher order oligomers in physiological buffer 24 and strikingly, into filaments at 106

high g-tubulin concentrations (Fig. S2C). Because γ-tubulins have been shown to arrange laterally, 107

as observed previously in its crystallized form28, a plus-ends outward orientation of g-tubulin 108

molecules could form a nucleation interface. 109

Surprisingly, the γ-tubulin oligomers efficiently nucleated MTs from αβ-tubulin subunits 110

(Fig. 2D and Movie S6) and even more strikingly, capped MT minus-ends while allowing newly 111

generated MT plus-ends to polymerize (Fig. 2E). This activity is similar to that of γ-TuRC, 112

suggesting that lateral γ-tubulin arrays on the nucleation interface of γ-TuRC are sufficient to 113

nucleate MTs. 114

Knowing that lateral γ-tubulin arrays in purified γ-tubulin oligomers and within g-TuRC 115

nucleate MTs, we hypothesized that the longitudinal affinity between γ-tubulin and αβ-tubulin at 116

the interface of γ-TuRC could be critical in regulating its nucleation efficiency. Using biolayer 117


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interferometry, we compared the interaction of ab-tubulin dimers with themselves versus with g-118

tubulin. Specific interactions between probe-bound αβ-tubulin and increasing concentrations of 119

unlabeled αβ-tubulin were measured (Fig. 2F), which must be longitudinal based on the observed 120

protofilaments in the ab-tubulin sample by EM (Fig. S2D). In contrast, no significant binding 121

between monomeric γ-tubulin and αβ-tubulin was detected (Fig. 2F), suggesting that the 122

heterogenous longitudinal affinity between g-tubulin and ab-tubulin on the nucleation interface 123

may be weaker compared to αβ-tubulin with another ab-tubulin molecule that occurs when the 124

MT lattice polymerizes. In sum, the difference in interaction strength is the basis for the kinetic 125

barrier we observed with g-TuRC but not with a blunt MT plus-end, which we summarize with an 126

interface interaction model (Fig. 2G). 127

We next asked how 3-4 tubulin dimers formed the rate-limiting species during γ-TuRC 128

nucleation. In stochastic simulations, the 13 available binding sites on g-tubulin molecules within 129

γ-TuRC were allowed to be occupied at random with αβ-tubulin subunits. We then assessed how 130

many ab-tubulin dimers need to assemble on g-TuRC to obtain two αβ-tubulin molecules on 131

neighboring sites and form a favorable configuration with a lateral contact between the two αβ-132

tubulins (Fig. 2H). The simulations show that 3.7 ± 1 tubulin dimers assemble on γ-TuRC to form 133

the first lateral contact between two αβ-tubulins (Fig. 2H), in striking agreement with the critical 134

nucleus size we measured. In sum, our data shows that a lateral γ-tubulin array positioned by γ-135

TuRC promotes MT nucleation. The low g-tubulin:ab-tubulin affinity requires binding of 3-4 αβ-136

tubulin dimers to g-TuRC to form the first lateral contact between two αβ-tubulin dimers and 137

overcome the kinetic barrier before entering the MT polymerization phase. This nucleation barrier, 138

in turn, provides the ability to further modulate MT nucleation via other factors. 139

140


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Regulation of γ-TuRC mediated nucleation by microtubule associated proteins 141

Recent work suggested that MT-associated proteins (MAPs), which stabilize or destabilize MT 142

plus-ends, influence MT nucleation in an analogous fashion7,22,23,27. We assessed this hypothesis 143

for MT nucleation by γ-TuRC. The protein TPX2 functions as an anti-catastrophe factor in vitro 144

22,23 and has been suggested to directly stimulate γ-TuRC-mediated nucleation21,29–31. Strikingly, 145

although TPX2 binds along the MT lattice, it does not increase nucleation activity of γ-TuRC (Fig. 146

3A and Movie S7). Similarly, the catastrophe factor EB1 does not decrease the nucleation activity 147

of γ-TuRC (Fig. S3A and Movie S8). Thus, in agreement with our previous results (Figs. 1 and 148

2A-B), destabilization of MT plus-ends and a competition between 149

polymerization/depolymerization is not sufficient to explain the properties of MT nucleation from 150

g-TuRC. Not surprisingly, decreasing the net rate of incorporation of tubulin into a MT using 151

Stathmin, which sequesters tubulin dimers32,33, or MCAK, which removes tubulin dimers from the 152

MT lattice and prevents polymerization34,35, decreased the number of MTs generated from γ-TuRC 153

(Fig. S3B). 154

155

How do γ-TuRC and XMAP215 synergistically nucleate microtubules? 156

At low tubulin concentration of 3.5 µM and 7 µM, where either none or very little MT nucleation 157

occurs from γ-TuRCs alone respectively, the addition of XMAP215 induced many surface-158

attached γ-TuRCs to nucleate MTs resulting in significant increase in MT nucleation rate (Fig. 3B-159

C and Movie S9). XMAP215 effectively decreases the minimal tubulin concentration necessary 160

for MT nucleation from γ-TuRC to 1.6 µM, which is very close to that needed for plus-end 161

polymerization. What is the sequence of events that leads to synergistic MT nucleation? By 162

directly visualizing γ-TuRC and XMAP215 molecules during the nucleation reaction, we found 163


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that XMAP215 and γ-TuRC molecules first formed a complex from which a MT was nucleated 164

(Fig. 3D and Movie S11). For 76% of the events (n=56), XMAP215 visibly persisted between 3 165

to over 300 seconds on γ-TuRC before MT nucleation, and with a 50% probability XMAP215 166

remained on the minus-end together with γ-TuRC (n=58). 167

Could XMAP215 accelerate nucleation by altering the critical tubulin nucleus that 168

assembles during γ-TuRC-mediated nucleation? Titrating tubulin at constant γ-TuRC and 169

XMAP215 concentrations (Fig. S4A and Movies S10) yielded a similar power-law dependence 170

between the MT nucleation rate and tubulin concentration (Fig. 3E). The resulting critical nucleus 171

size of 3.2 ± 1.2 is very similar to that for γ-TuRC alone (Fig. 3E). Moreover, the C-terminus of 172

XMAP215 (TOG5 and C-terminal domain), which directly interacts with γ-tubulin but not with 173

αβ-tubulin24, does not enhance MT nucleation from γ-TuRC (Fig. S4B). Altogether, γ-TuRC 174

determines the critical nucleus of ab-tubulin dimers for MT nucleation (Fig. 2H). XMAP215, 175

which directly binds to g-tubulin via its C-terminal domain, does not appear to activate g-TuRC 176

via a conformational change, but likely relies on N-terminal TOG domains to increase ab-tubulin 177

incorporation by effectively increasing the local ab-tubulin concentration, and thereby promoting 178

MT nucleation. 179

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Discussion 181

Decades after the discovery of ab-tubulin and MTs and the identification of γ-TuRC as the 182

universal MT nucleator17–19, it has remained poorly understood how MTs are being nucleated7,20,21. 183

Here, we show that γ-TuRC-mediated MT nucleation is more efficient than spontaneous MT 184

assembly, requiring fewer tubulin dimers to form the rate-limiting reaction intermediate. This 185

explains why MTs do not form spontaneously in the cell and why γ-TuRC is essential, addressing 186

a long debate on g-TuRC’s MT nucleation activity and requirement36–38. Spontaneous MT 187

assembly requires higher tubulin concentrations and occurs due to stronger longitudinally-188

interacting αβ/αβ-tubulin and weaker lateral interactions. In contrast, g-TuRC-mediated 189

nucleation, driven by the lateral adjacency of the g-tubulins on the nucleation interface, is sufficient 190

to overcome the intrinsically very weak ab-tubulin lateral interaction, thereby potentiating MT 191

nucleation. Thus, we propose that, in metazoans analogous to the S. cerevisiae γ-TuSC rings15,16, 192

GCPs within γ-TuRC restrict the number of laterally-arranged g-tubulin subunits, and position 193

them in the right geometry to template 13-pf MTs. Finally, our results show that 3-4 ab-tubulin 194

form the critical nucleus on g-TuRC, not 1 or 13 which would have been expected from previous 195

mechanistic hypotheses20. We find that on average 3-4 ab-tubulin dimers assemble on g-TuRC to 196

form the first lateral ab-/ab-tubulin contact and overcome the kinetic barrier that results from low 197

longitudinal affinity between g-:ab-tubulin on g-TuRC. However, alternative reaction 198

intermediates during nucleation from g-TuRC may exist. In the future, it will be important to 199

visualize the nucleation intermediates on g-TuRC, develop molecular simulations with 200

experimentally derived affinities at various interaction interfaces and evaluate whether additional 201

effects from tubulin straightening play a significant role in MT nucleation in the cell. 202


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The intermediate level of MT nucleation efficiency afforded by g-TuRC allows other 203

factors to further modulate its efficiency. As such, XMAP215 accelerates MT nucleation from γ-204

TuRC, while not altering the geometry of the ab-tubulin nucleus on g-TuRC or directly activating 205

g-TuRC. Future studies will be necessary to define the modes by which XMAP215 contributes to 206

g-TuRC-mediated MT nucleation, such as increasing the probability of the g/ab-tubulin interaction 207

or promoting straightening of incoming tubulin dimers. Our findings suggest that influencing g/ab-208

tubulin interaction favorably or unfavorably may underlie a dominant mechanism for regulating 209

nucleation in the cell by other, yet unidentified nucleation factors. Additionally, g-TuRC’s activity 210

is further regulated via accessory proteins such as CDK5RAP2, and NME72,20,39,40. While the 211

mechanisms of these additional regulation layers are yet to be defined, the insights on MT 212

nucleation by γ-TuRC and XMAP215 provide an essential basis to build upon. Finally, this work 213

opens the door to reconstitute cellular structures in vitro using MT nucleation from γ-214

TuRC/XMAP215 to further our understanding of how the cytoskeleton is generated to support cell 215

function. 216


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Supplementary Information 217

Supplementary Information includes four figures and ten videos. 218

219

Acknowledgements 220

We thank David Agard, Tim Mitchison, Michelle Moritz and Petry lab members for discussions. 221

This work was supported by an American Heart Association predoctoral fellowship 222

17PRE33660328 and a Princeton University Honorific Fellowship (both to AT), the NIH New 223

Innovator Award 1DP2GM123493, Pew Scholars Program in the Biomedical Sciences 00027340, 224

David and Lucile Packard Foundation 2014-40376 (all to SP), and the Center for the Physics of 225

Biological Function sponsored by the National Science Foundation grant PHY-1734030. 226

227

Author contributions 228

A.T. designed and performed research, analyzed the data and wrote the manuscript. S.P., J.W.S. 229

and H.A.S. supervised research and wrote the manuscript. 230

231

Competing financial interests 232

The authors declare no competing financial interests. 233

234

Abbreviations List 235

Microtubule (MT) 236

Microtubule associated protein (MAP) 237

Gamma-tubulin (γ-tubulin) and Gamma-tubulin ring complex (γ-TuRC) 238

Gamma-tubulin complex protein (GCP) 239


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Protofilament (pf) 240

Electron microscopy (EM) 241


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44. Reber, S. B. et al. XMAP215 activity sets spindle length by controlling the total mass of 340

spindle microtubules. Nat. Cell Biol. 15, 1116–1122 (2013). 341

45. Hannak, E. & Heald, R. Investigating mitotic spindle assembly and function in vitro using 342

Xenopus laevis egg extracts. Nat Protoc 1, 2305–2314 (2006). 343

46. Murray, A. W. & Kirschner, M. W. Cyclin synthesis drives the early embryonic cell cycle. 344

Nature 339, 275–280 (1989). 345

47. Bieling, P., Telley, I. A., Hentrich, C., Piehler, J. & Surrey, T. Fluorescence microscopy 346

assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, 347

and +TIP dynamics. Methods Cell Biol. 95, 555–580 (2010). 348

349


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Methods 350

351

Purification of recombinant proteins 352

C-terminal GFP was replaced with mCherry tag in the pET21a vector carrying EB141. Full-length 353

TPX2 with N-terminal Strep II-6xHis-GFP-TEV site tags was cloned into pST50Tr-354

STRHISNDHFR (pST50) vector42 using Gibson Assembly (New England Biolabs). N-terminal 355

6xHis-tagged, Xenopus laevis Stathmin 1A was a gift from Christiane Wiese (University of 356

Madison). N-terminal tagged 6xHis-TEV MCAK plasmid was a gift from Ryoma Ohi43. Wild-357

type XMAP215 with C-terminal GFP-7xHis plasmid was a gift from Simone Reber44 and was used 358

to clone XMAP215 with C-terminal SNAP-TEV-7xHis-StrepII tags, first into pST50 vector and 359

further into pFastBac1 vector. TOG5-CT truncation of XMAP215 was produced by cloning amino 360

acids 1091-2065 into pST50 vector with C-terminal GFP-7xHis-Strep tags. Human γ-tubulin TEV-361

Strep II-6xHis tags was codon-optimized for Sf9 expression, synthesized (Genscript), and further 362

cloned into pFastBac1 vector. 363

EB1, TPX2, Stathmin and XMAP215 TOG5-CT used in this study were expressed in E. 364

coli Rosetta2 cells (EMD Millipore) by inducing with 0.5-1 mM IPTG for 12-18 hours at 16°C or 365

7 hours at 25°C. Wild-type XMAP215, MCAK and γ-tubulin were expressed and purified from 366

Sf9 cells using Bac-to-Bac system (Invitrogen). The cells were lysed (EmulsiFlex, Avestin) and 367

E. coli lysate was clarified by centrifugation at 13,000 rpm in Fiberlite F21-8 rotor (ThermoFisher) 368

and Sf9 cell lysate at 50,000 rpm in Ti70 rotor (Beckman Coulter) for 30-45 minutes. 369

EB1 and Stathmin were purified using His-affinity (His-Trap HP, GE Healthcare) by first 370

binding in binding buffer (20mM NaPO4 pH 8.0, 500mM NaCl, 30mM Imidazole, 2.5mM PMSF, 371

6mM BME) and eluting with 300mM Imidazole, followed by gel filtration (HiLoad 16/600 372


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Superdex, GE Healthcare) into CSF-XB buffer (100mM KCl, 10mM K-HEPES, 5mM K-EGTA, 373

1mM MgCl2, 0.1mM CaCl2, pH 7.7 with 10% w/v sucrose). 374

TPX2 was first affinity purified using Ni-NTA beads in binding buffer (50mM Tris-HCl 375

pH 8.0, 750mM NaCl, 15mM Imidazole, 2.5mM PMSF, 6mM BME) and eluted with 200mM 376

Imidazole. All protein was pooled and diluted 4-fold to 200mM final NaCl. Nucleotides were 377

removed with a Heparin column (HiTrap Heparin HP, GE Healthcare) by binding protein in 378

250mM NaCl and isocratic elution in 750mM NaCl, all solutions prepared in Heparin buffer 379

(50mM Tris-HCl, pH 8.0, 2.5mM PMSF, 6mM BME). Peak fractions were pooled and loaded on 380

to Superdex 200 pg 16/600, and gel filtration was performed in CSF-XB buffer. 381

MCAK was first affinity purified by binding to His-Trap HP (GE Healthcare) in binding 382

buffer (50mM NaPO4, 500mM NaCl, 6mM BME, 0.1mM MgATP, 10mM Imidazole, 1mM 383

MgCl2, 2.5mM PMSF, 6mM BME, pH to 7.5), eluting with 300mM Imidazole, followed by gel-384

filtration (Superdex 200 10/300 GL, GE Healthcare) in storage buffer (10 mM K-HEPES pH 7.7, 385

300 mM KCl, 6mM BME, 0.1 mM MgATP, 1mM MgCl2, 10% w/v sucrose). 386

XMAP215-GFP was purified using His-affinity (His-Trap, GE Healthcare) by binding in 387

buffer (50mM NaPO4, 500mM NaCl, 20mM Imidazole, pH 8.0) and eluting in 500mM Imidazole. 388

Peak fractions were pooled and diluted 5-fold with 50mM Na-MES pH 6.6, bound to a cation-389

exchange column (Mono S 10/100 GL, GE Healthcare) with 50mM MES, 50mM NaCl, pH 6.6 390

and eluted with a salt-gradient up to 1M NaCl. Peak fractions were pooled and dialyzed into CSF-391

XB buffer. SNAP-tagged XMAP215 was first affinity purified with StrepTrap HP (GE Healthcare) 392

with binding buffer (50mM NaPO4, 270mM NaCl, 2mM MgCl2, 2.5mM PMSF, 6mM BME, pH 393

7.2), eluted with 2.5mM D-desthiobiotin, and cation-exchanged (Mono S 10/100 GL). Peak 394

fractions were pooled, concentrated and reacted with 2-molar excess SNAP-substrate Alexa-488 395


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dye (S9129, NEB) overnight at 4°C, followed by purification via gel filtration (Superdex 200 396

10/300 GL) in CSF-XB buffer. Approximately 70% labeling efficiency of the SNAP-tag was 397

achieved. 398

γ-tubulin was purified by binding to HisTrap HP (GE Healthcare) in binding buffer (50 399

mM KPO4 pH 8.0, 500 mM KCl, 1 mM MgCl2, 10% glycerol, 5mM Imidazole, 0.25 µM GTP, 5 400

mM BME, 2.5mM PMSF), washing first with 50 mM KPO4 pH 8.0, 300 mM KCl, 1 mM MgCl2, 401

10% glycerol, 25 mM imidazole, 0.25 µM GTP, 5 mM BME), and then with 50 mM K-MES pH 402

6.6, 500 mM KCl, 5mM MgCl2, 10% glycerol, 25 mM imidazole, 0.25 µM GTP, 5 mM BME) and 403

eluted in 50 mM K-MES pH 6.6, 500 mM KCl, 5mM MgCl2, 10% glycerol, 250 mM imidazole, 404

0.25 µM GTP, 5 mM BME. Peak fractions were further purified with gel filtration (Superdex 200 405

10/300 GL) in buffer 50 mM K-MES pH 6.6, 500 mM KCl, 5 mM MgCl2, 1 mM K-EGTA, 1 µM 406

GTP, 1 mM DTT. 407

All proteins were flash-frozen and stored at -80°C, and their concentration was determined 408

by analyzing a Coomassie-stained SDS-PAGE against known concentration of BSA (A7906, 409

Sigma). 410

411

Purification, biotinylated and fluorescent labeling of γ-TuRC 412

Endogenous γ-TuRC was purified from Xenopus egg extracts and labeled with the following steps 413

at 4°C. 7-8 ml of meiotic extract from Xenopus laevis eggs, prepared as described previously45,46, 414

was first diluted 5-fold with CSF-XBg buffer (10mM K-HEPES, 100mM KCl, 1mM MgCl2, 5mM 415

K-EGTA, 10% w/v sucrose, 1mM DTT, 1mM GTP, 10 µg/ml LPC protease inhibitors, pH 7.7), 416

centrifuged to remove large aggregates at 3500 rpm (Thermo Sorvall Legend XTR) for 10 minutes, 417

and the supernatant filtered sequentially with 1.2 µm and 0.8 µm Cellulose Acetate filters 418


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(Whatman) followed by 0.22 µm PES filter (ThermoFisher). γ-TuRC was precipitated by 419

incubating with 6.5% w/v PEG-8000k (Sigma) for 30 minutes and centrifuged at 17,000 rpm (SS-420

34 rotor, ThermoScientific) for 20 minutes. γ-TuRC-rich pellet was resuspended in CSF-XB buffer 421

with 0.05% v/v NP-40 using a mortar & pestel homogenizer, PEG was removed via centrifugation 422

at 136,000 xg for 7 minutes in TLA100.3 (Beckman Ultracentrifuge), and supernatant was pre-423

cleared by incubating with Protein A Sepharose beads (GE LifeSciences #17127901) for 20 424

minutes. Beads were removed, γ-TuRC was incubated with 4-5 mg of a polyclonal antibody 425

custom-made against C-terminal residues 413-451 of X. laevis γ-tubulin (Genscript) for 2 hours 426

on gentle rotisserie, and further incubated with 1ml washed Protein A Sepharose bead slurry for 2 427

hours. γ-TuRC-bound beads were washed sequentially with 30 ml of CSF-XBg buffer, 30 ml of 428

CSF-XBg buffer with 250 mM KCl (high salt wash), 10 ml CSF-XBg buffer with 5mM ATP 429

(removes heat-shock proteins), and finally 10 ml CSF-XBg buffer before labeling. For 430

biotinylation of γ-TuRC, beads were incubated with 25 µM NHS-PEG4-biotin (A39259, 431

ThermoFisher) in CSF-XBg buffer for 1 hour at 4°C, and unbound biotin was removed by washing 432

with 30 ml CSF-XBg buffer prior to elution step. For combined fluorescent and biotin labeling of 433

γ-TuRC, the wash step after ATP-wash consisted of 10 ml of labelling buffer (10mM K-HEPES, 434

100mM KCl, 1mM MgCl2, 5mM K-EGTA, 10% w/v sucrose, 0.5mM TCEP, 1mM GTP, 10 µg/ml 435

LPC, pH 7.2) and fluorescent labelling was performed by incubating the beads with 1 µM Alexa-436

568 C5 Maleimide (A20341, ThermoFisher). Unreacted dye was removed with 10 ml CSF-XBg 437

buffer, beads were incubated with 25 µM NHS-PEG4-biotin (A39259, ThermoFisher) in CSF-438

XBg buffer for 1 hour at 4°C, and unreacted biotin removed with 30 ml CSF-XBg buffer. Labeled 439

g-TuRC was eluted by incubating 2-3ml of g-tubulin peptide (residues 413-451) at 0.4mg/ml in 440

CSF-XBg buffer with beads overnight. After 10-12 hours, g-TuRC was collected by adding 1-2ml 441


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CSF-XBg buffer to the column, concentrated to 200 µl in 30k NMWL Amicon concentrator (EMD 442

Millipore) and layered onto a continuous 10-50 w/w % sucrose gradient prepared in a 2.2 ml ultra-443

clear tube (11x34 mm, Beckman Coulter) using a two-step program in Gradient Master 108 444

machine. Sucrose gradient fractionation of g-TuRC was performed by centrifugation at 200,000xg 445

in TLS55 rotor (Beckman Coulter) for 3 hours. The gradient was fractionated from the top in 11-446

12 fractions using wide-bore pipette tips and peak 2-3 fractions were identified by immunoblotting 447

against g-tubulin with GTU-88 antibody (Sigma). g-TuRC was concentrated to 80 µl in 30k 448

NMWL Amicon concentrator (EMD Millipore) and fresh purification was used immediately for 449

single molecule assays. Cryo-preservation of g-TuRC molecules resulted in loss of ring assembly 450

and activity. 451

452

Assessment of γ-TuRC with protein gel, immunoblot and negative stain electron microscopy 453

To assess the purity of g-TuRC, 3-5 µl of purified g-TuRC was visualized on an SDS-PAGE with 454

SYPRO Ruby stain (ThermoFisher) following the manufacturer’s protocol. Biotinylated subunits 455

of g-TuRC were assessed by immunoblotting with Streptavidin-conjugated alkaline phosphatase 456

(S921, ThermoFisher). g-TuRC purification was also assessed by visualizing using electron 457

microscopy. 4 µl of peak sucrose gradient fraction of g-TuRC was pipetted onto CF400-Cu grids 458

(Electron Microscopy Sciences), incubated at room temperature for 60 seconds and then wicked 459

away. 2% uranyl acetate was applied to the grids for 30 seconds, wicked away, and the grids were 460

air-dried for 10 minutes. The grids were imaged using Phillips CM100 TEM microscope at 64000x 461

magnification. 462

463

Preparation of functionalized coverslips 464


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22x22 mm, high precision coverslips (170±5 µm, Carl Zeiss, catalog # 474030-9020-000) were 465

functionalized for single molecule assays based on a recent protocol23,47 with specific 466

modifications. Briefly, coverslips were labelled on the surface to be functionalized by scratching 467

“C” on right, bottom corner, placed in Teflon racks, sonicated with 3N NaOH for 30 minutes, 468

rinsed with water and sonicated in piranha solution (2 parts of 30 w/w % hydrogen peroxide and 469

3 parts sulfuric acid) for 45 minutes. Coverslips were rinsed thrice in water, and all water was 470

removed by spin drying completely in a custom-made spin coater. Pairs of coverslips were made 471

to sandwich 3-glycidyloxypropyl trimethoxysilane (440167, Sigma) on the marked sides, placed 472

in glass petri dishes, and covalent reaction was performed in a lab oven at 75°C for 30 minutes. 473

Coverslips were incubated for 15 minutes at room temperature, the sandwiches were separated, 474

incubated in acetone for 15 minutes, then transferred to fresh acetone and quickly dried under 475

nitrogen stream. Coverslip sandwiches were prepared with a small pile of well mixed HO-PEG-476

NH2 and 10% biotin-CONH-PEG-NH2 (Rapp Polymere) in glass petri dishes, warmed to 75°C in 477

the lab oven until PEG melts, air bubbles were pressed out and PEG coupling was performed at 478

75°C overnight. The following day, individual coverslips were separated from sandwiches, 479

sonicated in MilliQ water for 30 minutes, washed further with water until no foaming is visible, 480

dried with a spin dryer, and stored at 4°C. Functionalized coverslips were used within 1 month of 481

preparation. 482

Imaging chambers were prepared by first assembling a channel on glass slide with double 483

sided tape strips (Tesa) 5 mm apart, coating the channel with 2mg/ml PLL(20)-g[3.5]- PEG(2) 484

(SuSOS) in dH2O, incubating for 20 minutes, rinsing out the unbound PEG molecules with dH2O 485

and drying the glass slide under the nitrogen stream. A piece of functionalized coverslip was cut 486


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with the diamond pen and assembled functionalized face down on imaging chamber. The prepared 487

chambers were stored at 4°C and used within a day of assembly. 488

489

Microtubule nucleation assay with purified γ-TuRC, microscopy and data analysis 490

The imaging channel was prepared as follows. First, 5% w/v Pluronic F-127 in dH2O was 491

introduced in the chamber (1 vol = 50 µl) and incubated for 10 minutes at room temperature. The 492

chamber was washed with 2 vols of assay buffer (80mM K-PIPES, 1mM MgCl2, 1mM EGTA, 493

30mM KCl, 0.075% w/v methylcellulose 4000 cp, 1% w/v D-(+)-glucose, 0.02% w/v Brij-35, 494

5mM BME, 1mM GTP) with 0.05 mg/ml κ-casien (casein buffer), followed by 1 vol of 0.5 mg/ml 495

NeutrAvidin (A2666, ThermoFisher) in casein buffer, incubated on a cold block for 3 minutes, 496

and washed with 2 vols of BRB80 (80mM K-PIPES, 1mM MgCl2, 1mM EGTA pH 6.8). 5-fold 497

dilution of g-TuRC in BRB80 was introduced in the flow chamber and incubated for 10 minutes. 498

Unattached g-TuRC molecules were washed with 1 vol of BRB80. 499

During the incubations, nucleation mix was prepared containing desired concentration of 500

αβ-tubulin (3.5-21 µM) purified from bovine brain with 5% Cy5-labeled tubulin along with 501

1mg/ml BSA (A7906, Sigma) in assay buffer, centrifuged for 12 minutes in TLA100 (Beckman 502

Coulter) to remove aggregates, a final 0.68 mg/ml glucose oxidase (SERVA, catalog # SE22778), 503

0.16 mg/ml catalase (Sigma, catalog # SRE0041) was added, and reaction mixture was introduced 504

into the flow chamber containing g-TuRC. 505

506

Total internal reflection fluorescence (TIRF) microscopy and analysis of microtubule 507

nucleation from γ-TuRC 508


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Nucleation of MTs was visualized with inverted Nikon TiE TIRF microscope using a 100X, 1.49 509

NA TIRF objective. An objective heater collar was attached (Bioptechs, model 150819-13) and 510

the temperature set-point of 33.5°C was used for experiments. Time-lapse videos were recorded 511

for 10 minutes at 0.5-1 frame per second using Andor iXon DU-897 camera with EM gain of 300 512

and exposure time of 50-200 ms each frame. Reference time-point zero (0 seconds) refers to when 513

the reaction was incubated at 33.5°C on the microscope, and for most reactions, imaging was 514

started within 30 seconds. 515

Growth speed of the plus-ends of MTs nucleated by g-TuRC was measured by generating 516

kymographs in ImageJ. Region of interest (ROI) for individual MTs were selected and resliced to 517

generate length-time plot, a line was fit to the growing MT, the slope of line represents growth 518

speed. The kinetics of MT nucleation from g-TuRC was measured as follows. A kymograph was 519

generated for every MT nucleated in the field of view. For most nucleation events, the time of 520

nucleation of the MT was obtained from observing the kymograph and manually recording the 521

initiation time point (see Fig. 1C for examples). For MTs where nucleation occurred before the 522

timelapse movie began or where the initiation was not clearly observed in the kymograph, the 523

shortest length of the MT that was clearly visible in the timelapse was measured and measured 524

average growth speed of MTs was used to estimate the time of nucleation. We verified that this 525

procedure accurately estimates the nucleation time for test case MTs where the nucleation event 526

was visible. The measurement of number of MTs (N(t)) nucleated versus time was generated from 527

a manual log containing the nucleation time for all MTs observed in the field of view, and a 528

representative set of curves is displayed in Fig. 1F. A straight line was fit to the initial (linear) 529

region of each N(t) versus t curve, rate of nucleation was obtained slope of each linear fit, and its 530

power-law relation with tubulin concentration was obtained and reported (Fig. 1G). 531


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532

Spontaneous microtubule nucleation and data analysis 533

Spontaneous MT assembly was visualized similar to g-TuRC-mediated nucleation with the 534

following changes. The pluronic, casein and NeutrAvidin incubations were performed identical to 535

g-TuRC nucleation assay but instead of attaching g-TuRCs, sucrose-based buffer (of the same 536

composition as used for g-TuRC elution) was diluted 5-fold with BRB80, introduced in the flow 537

chamber and incubated for 10 minutes. Washes were performed with 1 vol of BRB80, nucleation 538

mix was added, and imaging was performed as described above. MTs nucleate spontaneously in 539

solution fall down on the coverslip due to depletion forces during the 10 minutes of visualizing the 540

reaction. The number of MTs nucleated in the field of view were counted manually and plotted in 541

Fig. 1I. 542

543

Preparation and microtubule assembly from blunt microtubule seeds 544

Blunt MTs were prepared with GMPCPP nucleotide in two polymerization cycles as described 545

recently22. Briefly, a 50 µl reaction mixture was prepared with 20 µM bovine brain tubulin with 546

5% Alexa-568 labeled tubulin and 5% biotin-labeled tubulin, 1mM GMPCPP (Jena Bioscience) 547

in BRB80 buffer, incubated on ice for 5 minutes, then incubated on 37°C for 30 minutes to 548

polymerize MTs, and MTs were pelleted by centrifugation at 126,000 xg for 8 minutes at 30°C in 549

TLA100 (Beckman Coulter). Supernatant was discarded, MTs were resuspended in 80% original 550

volume of BRB80, incubated on ice for 20 minutes to depolymerize MTs, fresh GMPCPP was 551

added to final 1mM, incubated on ice for 5 minutes, a second cycle of polymerization was 552

performed by incubating the mixture at 37°C for 30 minutes, and MTs were pelleted again by 553

centrifugation. Supernatant was discarded and MTs were resuspended in 200 µl warm BRB80, 554


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29

flash frozen in liquid nitrogen in 5µl aliquots, stored at -80°C and found to be stable for months. 555

To verify that these MT seeds have blunt ends, frozen aliquots were quickly thawed at 37°C, 556

diluted 20-fold with warm BRB80, and incubated at room temperature for 30 minutes to ensure 557

blunt ends as described previously22. MTs were pipetted onto CF400-Cu grids (Electron 558

Microscopy Sciences), incubated at room temperature for 60 seconds and then wicked away. 2% 559

uranyl acetate was applied to the grids for 30 seconds, wicked away, and the grids were air-dried 560

for 10 minutes. The grids were imaged using Phillips CM100 TEM microscope at 130000 x 561

magnification and most MT ends were found to be blunt. 562

To assay MT assembly from blunt MT seeds, MT assembly experiments similar to g-TuRC 563

nucleation assays were performed with the following variation. A lower concentration 0.05 mg/ml 564

NeutrAvidin (A2666, ThermoFisher) was attached, and washes were performed with warm 565

BRB80 prior to attaching MTs. One aliquot of MT seeds was thawed quickly, diluted to 100-fold 566

with warm BRB80, incubated in the chamber for 5 minutes, unattached seeds were washed with 1 567

vol of warm BRB80, and the slide was incubated at room temperature for 30 minutes to ensure 568

blunt MT ends. Wide bore pipette tips were used for handling MT seeds to minimize the shear 569

forces that may result in breakage of MTs. Nucleation mix was prepared as described above and a 570

low αβ-tubulin concentration (1.4-8.7 µM) was used. MT assembly from blunt seeds was observed 571

immediately after incubating the slide on the objective heater. Imaging and analysis were 572

performed as described above for to g-TuRC nucleation assays. However, the probability curves 573

for MT assembly were obtained (Fig. 2B) by normalizing for the total number of seeds observed 574

in the field of view. Rate of assembly was plotted against [tubulin concentration – C*], where C* 575

represents the critical tubulin concentration below which MT ends do not polymerize obtained 576

directly from experimental measurements (Fig. S2A-B). 577


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578

Electron microscopy of γ-tubulin filaments in vitro 579

Purified γ-tubulin was observed to form higher order oligomers previously using analytical gel 580

filtration24. g-tubulin filaments were prepared by diluting pure g-tubulin to 1-5 µM to the buffer 581

50mM K-MES pH 6.6, 5mM MgCl2, 1mM EGTA, 100mM KCl. g-tubulin mixture were pipetted 582

onto CF400-Cu grids (Electron Microscopy Sciences), incubated at room temperature for 60 583

seconds and then wicked away. 2% uranyl acetate was applied to the grids for 30 seconds, wicked 584

away, and the grids were air-dried for 10 minutes. The grids were imaged using Phillips CM100 585

TEM microscope at 130000 x magnification and g-tubulin filaments were seen to form. At 500 586

mM KCl, g-tubulin filaments were not seen. 587

588

Nucleation of microtubules from purified γ-tubulin 589

MT assembly experiments from purified g-tubulin was performed similar to g-TuRC nucleation 590

assays described above with following variation. No avidin was attached to the coverslips, and 591

varying concentration of g-tubulin was prepared by diluting purified g-tubulin in a high salt buffer 592

(50mM K-MES pH 6.6, 500mM KCl, 5mM MgCl2, 1mM EGTA), centrifuging to remove 593

aggregates separately for 12 minutes in TLA100 before adding to the nucleation mix containing 594

15 µM αβ-tubulin (5% Cy5-labeled) with BSA, glucose oxidase and catalase as described above. 595

The reaction mixture was introduced into the flow chamber and imaged via TIRF microscopy. A 596

large number of MTs get nucleated immediately in the presence of 250 nM-1000 nM g-tubulin. 597

598

Measurement of affinity between purified γ-tubulin and αβ-tubulin 599


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Interaction assays between αβ-tubulin and g-tubulin were performed with biolayer interferometry 600

using Octet RED96e (ForteBio) instrument in an 8-channel plate format. The plate temperature 601

was held at 33°C and the protein samples were shaken at 400 rpm during the experiment. First, 602

Streptavidin or anti-His antibody coated biosensors (ForteBio) were rinsed in interaction buffer 603

(50mM K-MES pH 6.6, 100mM KCl, 5mM MgCl2, 1mM EGTA, 0.05% Tween20, 1mM GTP). 604

100 nM biotin-labeled αβ-tubulin, or blank buffer, was bound to Streptavidin sensor, or 200 nM 605

His-tagged g-tubulin to anti-His sensor until loaded protein results in a wavelength shift (Δλ) of 3 606

nm. Unbound protein was removed by rinsing the sensor in interaction buffer, and interaction with 607

αβ-tubulin was measured by incubating the sensor containing αβ-tubulin, g-tubulin or buffer with 608

0-35 µM unlabeled αβ-tubulin in interaction buffer for 5 minutes. Δλ (nm) was recorded as a 609

measure of the amount of unlabeled αβ-tubulin that binds to the sensor. Longitudinal interaction 610

occurs between αβ-tubulin dimers and the resulting protofilaments were verified by visualizing 611

the αβ-tubulin sample stained with 2% uranyl acetate using electron microscopy as described 612

above (Fig. S2D). 613

614

Simulation of site occupation on γ-TuRC by αβ-tubulin dimers 615

A simulation was performed in MATLAB for occupation of sites on g-TuRC by αβ-tubulin dimers. 616

A circular grid was simulated with 13 empty positions that were occupied one per unit time 617

stochastically such that a new position was selected by uniform random number generator and 618

filled. If a previously filled position was selected, a different position was selected by the random 619

number generator. The sequence in which the sites were occupied was followed. For each 620

simulation, the total number of sites that were occupied when the first two neighboring sites are 621


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32

filled was recorded. The simulation was repeated 10,000 times and the probability of occurrence 622

of first neighbor contact versus number of sites occupied is displayed in Fig. 2H. 623

624

Measuring the effect of microtubule associated proteins on γ-TuRC’s activity 625

Effect of microtubule associated proteins (MAPs) was measured on g-TuRC’s nucleation activity. 626

g-TuRC was attached on the coverslips using the setup described above and a control experiment 627

was performed with identical reaction conditions for each protein tested. Nucleation mix was 628

prepared containing 10.5 µM αβ-tubulin concentration (5% Cy5-labeled tubulin) as specified along 629

with 1mg/ml BSA and oxygen scavengers, and either buffer (control), 10nM GFP-TPX2, 100nM 630

EB1-mCherry, 5 µM Stathmin or 10nM MCAK was added. To test MCAK’s effect, the assay 631

buffer additionally contained 1mM ATP. The reaction mixture containing tubulin and MAP at 632

specified concentration was introduced into the flow chamber containing g-TuRC, and MT 633

nucleation was visualized by imaging the Cy5-fluorescent channel at 0.5-1 frames per second. For 634

TPX2 and EB1, fluorescence intensity of the protein was simultaneously acquired. The number of 635

MTs nucleated over time was measured as described above and the effect of protein on g-TuRC’s 636

nucleation activity was assessed by comparing nucleation curves with and without the MAP. 637

A similar set of experiments were performed to study the effect of XMAP215 on g-TuRC-638

mediated nucleation with the single molecule assays with the following differences. 20 nM of 639

XMAP215-GFP was added to nucleation mix prepared with 3.5-7 µM αβ-tubulin concentration 640

(5% Cy5-label) in XMAP assay buffer (80mM K-PIPES, 1mM MgCl2, 1mM EGTA, 30mM KCl, 641

0.075% w/v methylcellulose 4000 cp, 1% w/v D-(+)-glucose, 0.007% w/v Brij-35, 5mM BME, 642

1mM GTP). MTs nucleated from attached g-TuRC with and without XMAP215 were measured to 643

assess the efficiency of nucleation induced by XMAP215 (Fig. 3C). To assess if C-terminal of 644


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33

XMAP215 increases nucleation efficiency, wild-type XMAP215 was replaced with a C-terminal 645

construct of XMAP215: TOG5-Cterminus-GFP in the described experiment. 646

To measure the kinetics of cooperative nucleation XMAP215 and g-TuRC, a constant 647

density of g-TuRC was attached as described above and nucleation mix nucleation mix was 648

prepared with a range of αβ-tubulin concentration between 1.6-7 µM (5% Cy5-label) with 20 nM 649

of XMAP215-GFP in XMAP assay buffer, introduced into reaction chamber and MT nucleation 650

was imaged immediately by capturing dual color images of XMAP215 and tubulin intensity at 0.5 651

frames per second. 652

653

Triple-color imaging of XMAP215, γ-TuRC and microtubules 654

For triple-color fluorescence assays (Fig. 3D), Alexa-568 and biotin-conjugated γ-TuRC was first 655

attached to coverslips as described above with the following variation: 0.05 mg/ml of NeutrAvidin 656

was used for attaching γ-TuRC. Nucleation mix was prepared with 7 µM αβ-tubulin (5% Cy5-657

label), 10 nM Alexa-488 SNAP-tagged XMAP215 with BSA and oxygen scavengers in XMAP 658

assay buffer (80mM K-PIPES, 1mM MgCl2, 1mM EGTA, 30mM KCl, 0.075% w/v 659

methylcellulose 4000 cp, 1% w/v D-(+)-glucose, 0.007% w/v Brij-35, 5mM BME, 1mM GTP) 660

and introduced into the reaction chamber containing attached γ-TuRC. Three-color imaging per 661

frame was performed with sequential 488, 568 and 647 nm excitation and images were acquired 662

with EMCCD camera at 0.3 frames per second. 663


https://doi.org/10.1101/853010

34

Figure legends 664

665

Figure 1. Microtubule nucleation by γ-TuRC. 666

(A) Schematic of γ-TuRC mediated nucleation based on template model. (B) Purified, biotinylated 667

γ-TuRC molecules were attached and time-lapse of MT nucleation is shown. Arrows point to 668

nucleation sites. Representative kymographs of MTs nucleated from γ-TuRC are displayed in (C). 669

The experiment and analyses in (B-G) were repeated at least thrice with independent γ-TuRC 670

preparations. (D) Titrating tubulin concentration with constant the density of γ-TuRC. MT 671

nucleation from γ-TuRC begins at 7 µM tubulin. (E) MT plus-end growth speed increases linearly 672

with tubulin concentration. Linear fit (red line) with shaded 95% confidence intervals is displayed, 673

with critical concentration for polymerization as C* = 1.4 µM. Inset: Number of MTs nucleated 674

by γ-TuRCs within 120 seconds varies non-linearly with tubulin concentration. (F) Number of 675

MTs nucleated (N(t)) over time (t) is plotted for varying tubulin concentration to obtain rate of 676

nucleation as the slope of the initial part of the curves. (G) Number of tubulin dimers (n) in the 677

critical nucleus on γ-TuRC was obtained as 3.7 ± 0.5 from the equation !"!# $#→& = ()#*+, 678

displayed on a log-log plot. Data from two independent experiments was pooled and reported. (H) 679

Spontaneous MT nucleation (schematized) was measured with increasing tubulin concentration 680

and high concentrations. 14 µM tubulin is required. (I) Number of MTs (N(t=τ)) nucleated 681

spontaneously were plotted against tubulin concentration (Ctub). Power-law curve was fit as N(t=τ) 682

= k Ctubn and tubulin cooperativity (exponent) of n = 8 ± 1 was obtained. Experiments were 683

repeated twice independently with many supporting results and all data were pooled. Scale bars, 684

10 µm. See Figure S1 and Movies S1-S4. 685

686


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35

Figure 2. Molecular mechanism for γ-TuRC-mediated microtubule nucleation. 687

(A) Schematic and a micrograph of pre-formed, blunt MT seeds is shown and MT assembly from 688

them was observed (right) with varying tubulin concentration. (B) Cumulative probability of MT 689

assembly from seeds (p(t)) over time (t) is plotted and rate of nucleation was obtained as the slope 690

from initial part of the curves. (C) Tubulin dimers (n) needed for MT assembly from seeds was 691

from the relation !-!# $#→& = (()#*+ − )∗), displayed on a log-log plot. n = 1.2 ± 0.4 showing non-692

cooperative assembly of tubulin. (D) MTs nucleate from purified γ-tubulin oligomers efficiently 693

and (E) minus-ends of γ-tubulin-nucleated MTs remain capped while the plus-ends polymerize. 694

(F) Molecular interaction between γ/αβ-tubulin was probed with bio-layer interferometry. Buffer 695

(left), biotin-tagged αβ-tubulin (middle), or His-tagged γ-tubulin (right) were loaded on the probe 696

as bait and untagged αβ-tubulin at 0-35 µM as prey. Wavelength shift, Δ3 (nm) indicated no 697

binding between empty probe and αβ-tubulin or γ/αβ-tubulin, while that between αβ-/αβ-tubulin 698

was observed and confirmed to be longitudinal (protofilament-wise, Fig. S2D). (G) Interface 699

interaction model determines MT nucleation by γ-TuRC where lateral γ/γ-tubulin promote 700

nucleation while low γ/αβ-tubulin affinity tunes nucleation. (H) Simulations were conducted where 701

13 sites on γ-TuRC were stochastically occupied by αβ-tubulins. For two αβ-tubulin subunits to 702

form lateral bond by occupying neighboring sites, 3.7 ± 1 subunits bind on average on γ-TuRC, 703

predicting the size of critical nucleus. Experiments and analyses were repeated at least twice 704

independently with multiple supporting results. Scale bars, 10 µm. See Figure S2 and Movie S5-705

6. 706

707

Figure 3. Regulation of microtubule nucleation by TPX2 and XMAP215. 708


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36

(A) A constant density of γ-TuRC molecules were attached and 10.5µM tubulin ± 10nM GFP-709

TPX2 were added. MTs were counted (right plot) and TPX2 was did not affect γ-TuRC-mediated 710

nucleation. Scale bar, 10µm. (B) γ-TuRCs were attached and low concentration 3.5-7µM ± 20nM 711

XMAP215 was added. XMAP215 induces MT nucleation from γ-TuRC efficiently. (C) MT 712

nucleation events were counted and plotted. Scale bar, 10µm. (D) Sequence of events during 713

cooperative MT nucleation by γ-TuRC and XMAP215 was visualized using labeled γ-TuRC 714

(blue), XMAP215 (red) and tubulin (green). Time-lapse: γ-TuRC and XMAP215 form a complex 715

prior to MT nucleation. XMAP215 variably resides on γ-TuRC for long (>100 seconds, 716

kymograph 1) or short times (~3-10 seconds, kymograph 2) before MT nucleation and remains at 717

the minus-end with 50% probability. Scale bar, 5µm. (E) Titrating tubulin with constant γ-TuRC 718

and XMAP215 concentration. XMAP215/γ-TuRC nucleate MTs from 1.6 µM tubulin. Number of 719

MTs nucleated (N(t)) over time (t) is plotted (inset) and rate of nucleation was obtained. Tubulin 720

dimers (n) in critical nucleus was obtained as 3.2 ± 1.2 and displayed on a log-log plot. The 721

experiment was performed once for all concentrations denoted and supported by a number of 722

additional experiments. The remaining experiments were repeated more than twice with 723

independent γ-TuRC preparations with additional supporting results. See Figure S3-4 and Movies 724

S7-10. 725

726


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37

Supplementary Figure legends 727

728

Supplementary Figure 1. Controls for γ-TuRC-mediated and spontaneous microtubule 729

nucleation. 730

(A-B) Protein gel (left) of purified γ-TuRC was stained with SYPRO Ruby stain and biotinylated 731

sites on γ-TuRC visualized with alkaline phosphatase conjugated to avidin (right). Major, known 732

γ-TuRC components were detected in the purified protein and GCP2/3 are heavily biotinylated 733

during purification. Purified and biotinylated γ-TuRC was stained with uranyl acetate and 734

visualized with transmission electron microscopy. Scale bar, 100nm. The experiments were 735

repeated at least thrice with independent γ-TuRC preparations. 736

(C) Covalent-reaction of biotin with γ-TuRC does not affect the nucleation activity, as measured 737

by attaching γ-TuRC with anti-Mozart1 antibody and comparing the number of MTs nucleated by 738

untagged and biotinylated γ-TuRC. Scale bar, 10µm. 739

(D) Control reactions for γ-TuRC-mediated nucleation. MTs were nucleated by attaching purified 740

γ-TuRC (left), adding control buffer (middle) or missing avidin in the reaction sequence (right). 741

Robust MT nucleation only occurs with γ-TuRC attached to coverslips and not in control reactions. 742

Scale bar, 10µm. See Movie S2. 743

(E) MTs were first nucleated from γ-TuRC with Alexa 568-labeled tubulin (cyan), followed by 744

introduction of Cy5-labeled tubulin (magenta). New tubulin only incorporates on the freely 745

growing, plus-end but not at the nucleated minus-end. Scale bar, 10µm. The experiment was 746

performed more than three times. 747


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38

(F) Two representative kymographs of spontaneously nucleated MTs are displayed, demonstrating 748

that MTs grow from both the minus-end (dotted line) and the plus-end (solid line). Scale bar, 749

10µm. See Movie S4. 750

(G) MTs nucleation from γ-TuRCs or spontaneously were compared at two tubulin 751

concentrations: 10.5 µM and 14 µM. γ-TuRC nucleates 10-fold higher number of MTs than 752

spontaneous assembly. The experiment was performed twice with many supporting results. 753

See also Figure 1. 754

755

Supplementary Figure 2. Microtubule assembly from blunt seeds and filament formation by 756

purified γ-tubulin. 757

(A) MT assembly (magenta) from MT seeds with blunt ends (cyan) was assayed. Tubulin 758

concentration was titrated, and MT plus-end assembles starting from 2.45 µM tubulin, which is 759

only slightly above the critical concentration of polymerization. Scale bar, 10µm. 760

(B) Growth speed of MT plus-ends was measured from kymographs and critical concentration (C* 761

= 1.4 µM) was determined from the linear fit (red line) with shaded 95% confidence intervals. The 762

experiment and analyses in were repeated twice on independent days along with other supporting 763

data. See also Figure 2 and Movie S5. 764

(C) γ-tubulin self-assembles into filaments at high concentration and low-salt (100mM KCl) as 765

imaged with negative-stain electron microscopy, whereas γ-tubulin filaments were not observed 766

at high-salt (500mM KCl). Scale bar, 100nm. 767

(D) Transmission electron microscopy of bio-layer interferometry assay of Fig. 2F show that 768

protofilaments of αβ-tubulin form. The experiment was repeated twice. Scale bar, 100nm. 769

770


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39

Supplementary Figure 3. Effect of microtubule associated proteins on γ-TuRC-mediated 771

nucleation. 772

(A) γ-TuRC molecules were attached to coverslips and either tubulin alone (pseudo-colored as 773

magenta, left) or tubulin with 100nM EB1-mCherry (pseudo-colored as cyan, right) was added to 774

the reaction. Number of MTs nucleated were measured (right plot) and EB1 was observed to 775

neither increase nor decrease γ-TuRC-mediation nucleation despite functioning as a catastrophe 776

factor in vitro. The experiment was repeated at least twice with independent γ-TuRC preparation. 777

See also Movie S8. Scale bar, 10µm. 778

(B) γ-TuRC molecules were attached to coverslips and either tubulin alone (left images), tubulin 779

with 10nM MCAK (top right) or tubulin with 5µM Stathmin (bottom right) was added to the 780

reaction. Both MCAK and Stathmin were observed to decrease the number of MTs nucleated 781

because of their role in decreasing the net polymerization of a MT. The experiment was repeated 782

at least twice with independent γ-TuRC preparations. Scale bar, 10µm. 783

784

Supplementary Figure 4. Cooperative microtubule nucleation XMAP215 and γ-TuRC. 785

(A) γ-TuRC molecules were attached and increasing concentration of tubulin was added with 786

20nM XMAP215. XMAP215 was found to induce MT nucleation from γ-TuRC molecules at even 787

low tubulin concentration of 1.6-3.5 µM where γ-TuRCs alone do not nucleate MTs. See Figure 788

3E. Scale bar, 10µm. 789

(B) The role of C-terminal region of XMAP215 was tested in cooperative nucleation with γ-TuRC. 790

MTs nucleated by γ-TuRC alone (left), γ-TuRC with 20nM full-length XMAP215 (middle) or γ-791

TuRC with 20nM C-terminal domain of XMAP215 were visualized. The C-terminal domains of 792


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40

XMAP215 do not stimulate MT nucleation from γ-TuRC. The experiment was repeated twice with 793

independent γ-TuRC preparations. Scale bar, 10µm. 794

795


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41

Movie Legends 796

797

Movie 1. Microtubule nucleation from γ-TuRC complexes 798

γ-TuRC was attached to functionalized coverslips and MT nucleation was observed upon 799

introducing fluorescent αβ-tubulin (gray). MTs nucleated from individual γ-TuRC molecules from 800

zero length at 15µM αβ-tubulin and the plus-end of nucleated MTs polymerized, but not its minus-801

end. Elapsed time is shown in seconds, where time-point zero represents the start of reaction. Scale 802

bar, 10 µm. 803

804

Movie 2. Microtubule nucleation from γ-TuRC is specific 805

γ-TuRC was immobilized on coverslips (leftmost panel) and MT nucleation was observed upon 806

introducing fluorescent αβ-tubulin (gray). Control reactions where either no γ-TuRC was added 807

(middle panel) or γ-TuRC was not specifically attached (rightmost panel) did not result in MT 808

nucleation. Elapsed time is shown in seconds, where time-point zero represents the start of 809

reaction. Scale bar, 10 µm. 810

811

Movie 3. γ-TuRC molecules nucleated microtubules efficiently 812

Constant density of γ-TuRC was attached while concentration of fluorescent αβ-tubulin was 813

titrated (3.5-21µM) and MT nucleation was observed. γ-TuRC molecules nucleated MTs starting 814

from 7µM tubulin and MT nucleation increased non-linearly with increasing tubulin concentration. 815

Elapsed time is shown in seconds, where time-point zero represents the start of reaction. Scale bar, 816

10 µm. 817

818


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42

Movie 4. Spontaneous microtubule nucleation occurs at high tubulin concentration 819

Concentration of fluorescent αβ-tubulin was titrated (7-21µM) and spontaneous MT nucleation 820

was assayed. MTs nucleated spontaneously starting from high concentration of 14µM tubulin and 821

MT nucleation increased non-linearly with tubulin concentration. Both plus- and minus-ends of 822

the assembled MTs polymerize. Elapsed time is shown in seconds, where time-point zero 823

represents the start of reaction. Scale bar, 10 µm. 824

825

Movie 5. Microtubule assembly from blunt plus-ends resembles polymerization 826

MTs with blunt ends (seeds, cyan) were generated and attached to functionalized coverslips. 827

Varying concentration of fluorescent αβ-tubulin was added (1.4-8.7µM, pseudo-colored as 828

magenta) and MT assembly from seeds was assayed. MTs assembled at concentration above 829

1.4µM tubulin, which is the minimum concentration needed for polymerization of MT plus-ends 830

(C*). MT assembly from seeds increased linearly with the concentration of assembly-competent 831

tubulin (C-C*). Elapsed time is shown in seconds, where time-point zero represents the start of 832

reaction. Scale bar, 10 µm. 833

834

Movie 6. Arrays of purified γ-tubulin nucleate microtubules 835

Purified γ-tubulin nucleated MTs. Fluorescent αβ-tubulin (10.5µM, colored as gray) was added to 836

purified γ-tubulin at increasing concentration, and MT nucleation was assessed. MTs assembled 837

from 250-1000 nM γ-tubulin, where γ-tubulin alone self-assembled into higher order oligomers 838

and filaments in lateral γ/γ-tubulin arrays. Minus-ends of γ-tubulin-nucleated MTs did not 839

polymerize, while the plus-ends did. Elapsed time is shown in seconds, where time-point zero 840

represents the start of reaction. Scale bar, 10 µm. 841


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43

842

Movie 7. TPX2 does not increase γ-TuRC’s microtubule nucleation activity 843

γ-TuRC was immobilized on coverslips and MT nucleation was observed upon introducing 844

fluorescent αβ-tubulin (10.5µM, pseudo-colored as magenta) without or with 10nM GFP-TPX2 845

(pseudo-colored as cyan) in the left and right panels respectively. TPX2 bound along the nucleated 846

MTs but did not increase the MT nucleation activity of γ-TuRC molecules. Elapsed time is shown 847

in seconds, where time-point zero represents the start of reaction. Scale bar, 10 µm. 848

849

Movie 8. EB1 does not decrease the microtubule nucleation activity of γ-TuRC 850

γ-TuRC was immobilized on coverslips and MT nucleation was observed upon introducing 851

fluorescent αβ-tubulin (10.5µM, pseudo-colored as magenta) without or with 100nM EB1-852

mCherry (pseudo-colored as cyan) in the left and right panels respectively. EB1 binds the plus-853

ends of nucleated MTs but did not decrease the MT nucleation activity of γ-TuRC molecules. 854

Elapsed time is shown in seconds, where time-point zero represents the start of reaction. Scale bar, 855

10 µm. 856

857

Movie 9. XMAP215 increases microtubule nucleation activity of γ-TuRC 858

γ-TuRC was immobilized on coverslips and MT nucleation was assayed with low concentratio

Molecular mechanism of microtubule nucleation from gamma ...61 TuRC (Fig. S1E), we showed that γ-TuRC caps the MT minus-end, while only the plus-end 62 polymerizes. Altogether, our

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